Metagenomic insights into the wastewater resistome before and after purification at large‑scale wastewater treatment plants in the Moscow city

Wastewater treatment plants (WWTPs) are considered to be hotspots for the spread of antibiotic resistance genes (ARGs). We performed a metagenomic analysis of the raw wastewater, activated sludge and treated wastewater from two large WWTPs responsible for the treatment of urban wastewater in Moscow, Russia. In untreated wastewater, several hundred ARGs that could confer resistance to most commonly used classes of antibiotics were found. WWTPs employed a nitrification/denitrification or an anaerobic/anoxic/oxic process and enabled efficient removal of organic matter, nitrogen and phosphorus, as well as fecal microbiota. The resistome constituted about 0.05% of the whole metagenome, and after water treatment its share decreased by 3–4 times. The resistomes were dominated by ARGs encoding resistance to beta-lactams, macrolides, aminoglycosides, tetracyclines, quaternary ammonium compounds, and sulfonamides. ARGs for macrolides and tetracyclines were removed more efficiently than beta-lactamases, especially ampC, the most abundant ARG in the treated effluent. The removal efficiency of particular ARGs was impacted by the treatment technology. Metagenome-assembled genomes of multidrug-resistant strains were assembled both for the influent and the treated effluent. Ccomparison of resistomes from WWTPs in Moscow and around the world suggested that the abundance and content of ARGs depend on social, economic, medical, and environmental factors.

The Lyuberetskiy WWTP complex of JSC "Mosvodokanal" carry out the treatment of wastewater in the city of Moscow with a capacity of about 2 million m 3 per day.This complex consists of several wastewater treatment units (hereafter referred to as WWTPs).They purify the same inflow wastewater but otherwise are independent installations between which there is no transfer and mixing of AS.Two WWTPs implementing different technologies for wastewater treatment were chosen as the objects of study.The first one (LOS) is operated using anaerobic/anoxic/oxic process, also known as the University of Cape Town (UCT) technology.There the sludge mixture first enters the anaerobic zone, where phosphate-accumulating microorganisms (PAO) consume easily degradable organics, then to the anoxic zone, where denitrification and accumulation of phosphates by denitrifying PAO occur, and finally to the aerobic zone, where organic matter and ammonium are oxidized while PAO

DNA isolation, 16S rRNA gene sequencing and analysis
Total genomic DNA was isolated using a Power Soil DNA isolation kit (Qiagen, Germany).DNA for each sample was isolated in four parallel replicates, which were then pooled.PCR amplification of 16S rRNA gene fragments comprising the V3-V4 variable regions was performed using the universal primers 341F (5′-CCT AYG GGDBGCWSCAG) and 806R (5′-GGA CTA CNVGGG THTCT AAT ) 20 .The obtained PCR fragments were bar-coded and sequenced on Illumina MiSeq (2 × 300 nt reads).Pairwise overlapping reads were merged using FLASH v.1.2.11 21 .All sequences were clustered into operational taxonomic units (OTUs) at 97% identity using the USEARCH v.11 program 22 .Low quality reads were removed prior to clustering, chimeric sequences and singletons were removed during clustering by the USEARCH algorithms.To calculate OTU abundances, all reads obtained for a given sample were mapped to OTU sequences at a 97% global identity threshold by USEARCH.The taxonomic assignment of OTUs was performed by searching against the SILVA v.138 rRNA sequence database using the VSEARCH v. 2.14.1 algorithm 23 .
The diversity indices at a 97% OTU cut-off level were calculated using USEARCH v.11 22 .To avoid sequencing depth bias, the numbers of reads for each sample were randomly sub-sampled to the size of the smallest set.

Efficiency of wastewater treatment
Two wastewater treatment technologies were used in the investigated WWTPs,-nitrification/denitrification at NLOS2 and more advanced anaerobic/anoxic/oxic UCT process at LOS. LOS removed more than 99.5% of organic matter (according to the BOD5 data) and more than 99.9% of ammonium while the performance of NLOS2 was poorer (Table 1).Particularly noticeable differences were observed in nitrate and nitrite concentrations in the effluents suggesting the lower efficiency of denitrification in the NLOS2.Interestingly, although the NLOS2 unit was not designed to remove phosphorus, the concentration of phosphates in the treated effluent at this WWTP is only slightly higher than at LOS.The treated influent at LOS contains fewer solids consistently with lower SVI.Overall, the technology used at LOS plant is more efficient.
Activated sludge of WWTP bioreactors is a complex microbial community consisting of physiologically and phylogenetically heterogeneous groups of microorganisms involved in the removal of major contaminants from wastewater.The composition of AS microbiomes was very different from the microbiome of incoming wastewater (Fig. 1).The phyla Campylobacterota (less than 0.5%) and Firmicutes (2-4%) were much less abundant in AS microbiomes.Proteobacteria was the dominant group in the microbiomes of AS (23-40%), but its composition differed from the microbiome of influent wastewater: instead of the fecal microflora (Enterobacterales and others) the AS community harbored lineages involved in the purification processes (Competibacteraceae, Rhodocyclaceae, Nitrosomonadaceae, etc.).Likewise, Bacteroidota were among the most numerous phyla in AS microbiomes at both LOS (6.5%) and NLOS2 (14.1%), but instead of Bacteroidales mostly comprised Chitinophagales and Sphingobacteriales typical for AS communities.The numerous groups of AS community also included Chloroflexi (22% and 10% in LOS and NLOS2, respectively), Patescibacteria (1.8% and 9.9%), Nanoarchaeota (4.3% and 9.1%), Nitrospirota (3.9% and 7.3%), Verrucomicrobiota and Myxococcota (about 4% in both WWTPs).Bacteria that play an important role in the removal of nitrogen (Nitrospira and Nitrosomonas) and phosphorus (Dechloromonas), as well as glycogen-accumulating Ca.Competibacter, have been found in large numbers.The abundance of these functional groups is consistent with the high efficiency of nitrogen and phosphorus removal.
The main source of microorganisms in treated effluent is the AS, from which they are washed out; bacteria from the influent water may also be present in minor amounts.Therefore, as expected, the microbiome  composition of treated wastewater was similar to that of activated sludge.Consistently, compositions of microbiomes of treated effluent were similar to that of AS samples.However, some differences were observed, in particular, the microbiomes of the treated effluent contained many Cyanobacteria (7.74% and 3.49% for LOS and NLOS2, respectively) which were found in minor amounts both in the influent water and in the ASs (< 0.5%).
Probably, these light-dependent bacteria proliferate in the final clarifier and then can be easily washed out with the effluent.
About twice less ARGs were identified in AS samples from both WWTPs.Like in the influent, beta-lactamases of classes A, D, and metallo-beta-lactamases were the most numerous, while only a few genes for class C enzymes were found (Table 2).Other families of ARGs, numerous in the influent, were also numerous in AS microbiomes.A notable difference between the resistomes of the AS samples is the greater number of rifampin-ADP-ribosyltransferase genes (arr) in NLOS2 compared to LOS (63 vs 33).The largest number of arr genes was assigned to Bacteroidota, and the lower relative abundance of this phylum in AS at LOS likely explains these differences.Like in the wastewater, resistance to beta-lactams, macrolides, rifamycin, aminoglycosides, and tetracyclines was the most common (Fig. 2).On the contrary, genes for some drug classes were underrepresented in AS resistomes, especially for diaminopyrimidines (3 and 2 genes for LOS and NLOS2, respectively) and glycopeptide antibiotics (2 and 0 genes).
The results of metagenomic analysis of treated effluent showed that the diversity of these resistomes was only slightly higher than that of the corresponding AS samples.This result was expected since the main source of microorganisms in the effluent is activated sludge, from which they are partially washed.However, resistomes of treated effluent at both WWTPs contains about twice more class A beta-lactamase genes than AS samples suggesting less efficient absorption of their host bacteria at AS particles (Table 2).

Quantitative analysis of antibiotic resistance genes of WWTP
The results described above provide information on the diversity of resistance genes, but not on their abundance in the metagenomes, which depends on the abundance of corresponding bacterial hosts.To quantify the Table 2. Resistomes of wastewater and AS samples, classified by gene families.www.nature.com/scientificreports/ shares of individual ARGs in the metagenome and resistome, the amounts of metagenomic reads mapped to the corresponding ARGs in contigs were determined.In total, the resistome accounted for about 0.05% of the metagenome of wastewater supplied for treatment, while the shares of resistomes in the metagenomes of AS and treated effluent samples were 0.02% and 0.014% at the LOS and NLOS2 WWTPs, respectively.Quantitative analysis of the content of individual ARGs in metagenomes showed that the structure of the influent resistome was very different from that of AS and treated effluent.The relative content of ARGs accounting for more than 1% in at least one analyzed resistome is shown in Fig. 3.The LOS and NLOS2 WWTPs differed significantly from each other, and the differences between the AS and effluent resistomes at each WWTP were much less pronounced.

Number of ARGs INFL AS-LOS CW-LOS AS-NLOS2 CW-NLOS2
The resistome of the influent was not only the most diverse, but also the most even in composition.The shares of none of the ARGs exceeded 5% of the resistome, and the 23 most common ARGs accounted for a half of the resistome.The most abundant ten ARGs were qacE, sul1, ampC, blaOXA, msr(E), erm(B), mph(E), tet(C), aph(3'')-Ib and aph( 6)-Id, conferring resistance to antiseptics, sulfonamides, beta-lactams, macrolides, aminoglycosides (streptomycin), and tetracyclines.
AS and treated effluent at LOS plant was strongly dominated by a single AGR type, class C beta-lactamase ampC, accounting for about 45% of their resistomes.This gene was also the most abundant one in the resistomes of AS and effluent at NLOS2 (14.8% and 18.2%, respectively).Apparently it originates from the  www.nature.com/scientificreports/influent wastewater supplied for treatment where its share in the resistome was 3.2%.AmpC β-lactamases are considered clinically important cephalosporinases encoded on the chromosomes and plasmids of various bacteria (especially Enterobacteriaceae), where they mediate resistance to cephalothin, cefazolin, cefoxitin and most penicillins 35 .Close homologues of this gene, with a nucleotide sequence identity of 99.8-100%, have been found in plasmids and chromosomes of various Proteobacteria (Thauera, Sphingobium, Aeromonas etc.).Since in all samples ampC was found in short contigs with very high coverage, it is likely widespread in the genomes of various bacteria in different genetic contexts.
The second most abundant ARG in the resistomes of AS samples was sulfonamide-resistant dihydropteroate synthase (sul1).It accounted for 4-5% of AS and treated effluent resistomes in LOS and for about 11% in NLOS2, while its share in the influent water resistome was about 5%.The sul1 gene is usually found in class 1 integrons being linked to other resistance genes, including qacE 36 .Consistently, sul1 and qacE were found in one contig assembled for the influent water samples and assigned to Gammaproteobacteria. Another sulfonamide-resistance gene, sul2, was also numerous, accounting for about 2% of the resistomes in the influent and LOS samples, and for about 4% in the AS and water treated at NLOS2.
Since ARGs entering the activated sludge and then into the treated effluent originate mostly from wastewater supplied for treatment, the absolute majority of ARGs present in the influent in significant amounts (more than 0.2% resistome) in were also found in AS and effluent samples.The only exception macrolide 2′-phosphotransferase gene mph(B) accounting for 0.51% in the influent resistome.Likewise, all ARGs accounting for more than 0.2% of resistomes in the treated effluent were present also in the influent.

Potential multidrug resistant strains
One of the most important public health problems is the spread of multidrug resistant pathogens (MDR), which refers to resistance to at least one agent in three or more chemical classes of antibiotic (e.g. a beta-lactam, an aminoglycoside, a macrolide) 37 .Such strains can arrive with wastewater entering the treatment, and also form in AS communities.AS are dense and highly competitive microbial communities, which, along with the presence of sublethal concentrations of antibiotics and other toxicants in wastewater, creates ideal conditions not only for the selection of resistant strains, but also for the formation of multiple resistance through horizontal gene transfer 4 .To identify MDR bacteria, we binned metagenomic contigs into metagenome-assembled genomes (MAGs) and looked for MAGs comprising several ARGs.Only MAGs with more than 70% completeness and less than 15% contamination were selected for analysis: 117, 56, 72, 94 and 121 for influent, AS of LOS, effluent of LOS, AS of NLOS2 and effluent of NLOS2, respectively.Five MAGs of MDR bacteria were identified in the metagenome of the influent, one-in AS of LOS, two-in the LOS effluent and one in the NLOS2 effluent (Table 3).These MAGs were assigned to unclassified genus-level lineages of Ruminococcaceae and Cyclobacteriaceae, Phocaeicola vulgatus, Streptococcus parasuis, Ancrocorticia sp., Enterococcus sp., Bacillus cereus and Undibacterium sp.

Disscussion
We characterized the composition of microbial communities and the resistomes of influent wastewater, activated sludge and treated effluent from two WWTPs in city of Moscow, where various biological water treatment technologies are used.Among the predominant bacteria in the influent wastewater we found mainly fecal contaminants of the genera Collinsella, Bacteroides, Prevotella, Arcobacter, Arcobacteraceae, Blautia, Faecalibacterium, Streptococcus, Acinetobacter, Aeromonas and Veillonella [38][39][40][41][42][43] .Previously, we performed 16S rRNA gene profiling of wastewater before and after treatment at one WWTP (LOS) and revealed that all abovementioned potential pathogens were efficiently removed and their relative abundance in the water microbiome decreased by 50-100 times 44 .Similar pattern of removal of potential pathogenic bacteria was observed here for NLOS2 where another water treatment technology is used.
An important indicator of the dissemination of ARG is the proportion of the resistome in the entire metagenome before and after wastewater treatment.In the influent, the resistome accounted for about 0.05% of the metagenome, which corresponds to approximately two ARGs per bacterial genome.Approximately the same values are typical for most countries 3 .After treatment, the fraction of the resistome in the wastewater metagenomes decreases, but, surprisingly, only by 2-4 times.However, since the total concentration of microorganisms in treated effluent is approximately two orders of magnitude lower than in raw wastewater, it is likely that the total abundance of ARGs in the treated effluent is significantly reduced.
Apparently, fecal contaminants effectively removed during treatment are not the only carriers of ARG in wastewater, which are also found in bacteria characteristic of activated sludge and thus appearing in the effluents.Unfortunately, due to the high diversity of microbiomes and the tendency of ARG to be present in multiple copies in different genomic environments, most of the contigs containing ARG turned out to be short, which did not allow to define their taxonomic affiliation.
The resistome of influent water includes 26 ARGs, the share of which is more than 1%.Among of them the prevalence of ampC, aadA, qacE, bla, qacF and qacL is specific for Moscow WWTPs, since these genes were not among the 50 most common ARGs according to the results of a worldwide analysis of wastewater resistomes in large cities 3 .Different ARGs were most "evenly" represented in the influent wastewater while in the AS and treated effluent, a clear selection of particular types of ARGs was observed, which obviously reflects a change in the composition of microbiomes.A vivid example is the increase in the proportion of ampC in the resistomes, especially at LOS.
The discovered ARGs can confer resistance to most classes of antibiotics and among the resistomes of the studied WWTPs in the city of Moscow, genes conferring resistance to beta-lactam antibiotics were the most common, they accounted for about 26% of the resistome in the water supplied for treatment (Fig. 4).Similar values have been observed for wastewater in some other countries, particularly in Eastern Europe and Brazil, where 20 to 25% of reads were assigned to ARGs conferring resistance to beta-lactams 3 .According to data for 2021, beta lactams accounted for about 40% of the total antibiotic consumption in Russia in the medical sector 45 .
Like in most wastewater resistomes in different countries, ARGs conferring resistance to macrolides, aminoglycosides and tetracycline were also among the most abundant in wastewater from Moscow (Fig. 4).Resistance to macrolides, rather than beta-lactams, was most common in wastewater from most countries in Europe and North America, while in Moscow ARGs to macrolide were the second most common.Macrolides and tetracyclines are also widely used in medicine in Russia (20% and 5% of total antibiotic consumption in 2021, respectively).On the contrary, medical consumption of aminoglycosides in Russia is rather low (< 1% of the total), therefore, the high abundance of relevant ARGs was unexpected.The opposite pattern was observed for quinolones, which make up about 22% of the antibiotics used in medicine, but their ARGs accounted for only about 1% of the resistome.However the main mechanisms of resistance to quinolones, mutations in the target enzymes, DNA gyrase and DNA topoisomerase IV, and increased drug efflux 46 , were not addressed in our study.
A peculiar feature of Moscow wastewater resistome was the high content of resistance genes to sulfonamides (about 9%), which were not among the major genes in wastewater resistomes worldwide 3 .Sulfonamides are synthetic antimicrobial agents that currently have limited use in the human medicine, alone or mainly in combination with trimethoprim (a dihydrofolate reductase inhibitor), in the treatment of uncomplicated respiratory, urinary tract and chlamydia infections 7,47 .Different sulfonamide ARGs (sul1, sul2 and sul3) were detected in the wastewater in the some countries, including Denmark, Canada, Spain and China, applying culture dependent, independent and qPCR methods 7 .The opposite picture was observed for streptogramin resistance genes, which were among the ARGs in the majority of resistomes worldwide, but in Moscow wastewater they accounted for less than 1%.This is probably due to the limited use of this drug in Russia.
Another distinguishing feature of the resistome of wastewater in Moscow is the high content of ARGs conferring resistance to quaternary ammonium compounds (QAC), about 9%.It can be explained by the frequent use of these antiseptics in medicine.QACs are active ingredients in more than 200 disinfectants currently recommended for inactivation the SARS-CoV-2 (COVID-19) virus 48 .A recent study showed that the number of QACs used to inactivate the virus in public facilities, hospitals and households increased during the COVID-19 pandemic 49 .Indeed, the results of a study dedicated to the study of wastewater resistome worldwide 3 did not reveal the presence of QAC ARGs in the wastewater, since the samples for this study were collected before the pandemic.
An important issue is the extent to which different water treatment technologies remove ARGs.The effective removal of ARG was primary due to a decrease in the concentration of microorganisms in treated effluent, since the share of resistome in the metagenome after treatment decreased by only 2.6 -3.7 times and the NLOS2 plant appeared to be more effective in this respect.However, compared to LOS, treated effluent at NLOS2 contains approximately twice as much suspended solids, probably due to poorer settling characteristics of the sludge indicated by the higher SVI.Therefore, the overall efficiency of removing ARGs from wastewater at two WWTPs may be similar.
Considering the relative abundances of ARGs in the resistomes, genes conferring resistance to macrolides and tetracyclines were removed more efficiently than beta lactamases, especially ampC, and rifampin ADPribosyltransferase genes.The low efficiency of removal of the ampC gene and the increase in its abundance in the resistome after wastewater treatment were previously reported for WWTPs in Germany 50 .Efficient removal of ARGs to macrolides (ermB, ermF, mph(A), mef(A)) and tetracyclines (tet(A), tet(C), tet(Q), tet(W)) has been reported in a number of studies worldwide 51 .ARGs enabling resistance to sulfonamides, tetracyclines and chloramphenicol were more efficiently removed at LOS than at NLOS2, while the opposite was observed for beta lactamases (Fig. 4).The later became the most abundant class of ARGs in the treated effluent.
Metagenomic analysis not only identified resistance genes, but also revealed probable MDR strains based on the analysis of assembled MAGs.We identified 9 such strains in both influent, AS and treated effluent.The real number of MDR strains is probably higher, since only a small fraction of all metagenomic contigs was included in the assembled high quality MAGs.
Phocaeicola vulgatus, (formerly Bacteroides vulgatus), is a mutualistic anaerobic bacteria commonly found in the human gut microbiome and frequently involved in human infections.The results of whole genome analysis showed presence of blaTEM-1 and blaCMY-2 ARGs, which confers resistant to beta-lactams 52,53 .P. vulgatus was also identified as potential host for the transmission of tetracycline ARGs 54 .Streptococcus parasuis is an important zoonotic pathogen that causes primarily meningitis, sepsis, endocarditis, arthritis, and pneumonia in both pigs and humans 55 .A variety of MDR strains of this bacterium have been described.For instance, S. parasuis strain H35 was isolated from a lung sample of a pig in China; several ARGs, including optrA, catQ, erm(B), lsa(E), msr(D), mef(A), mdt(A), tet(M), lnu(B), aadE and two copies of aacA-aphD, were found in the chromosome and cfr(D) was detected on plasmid pH35-cfrD 56 .MDR strain of Bacillus cereus was identified in the effluent water microbiome.This bacterium is known as human pathogen and a common cause of food poisoning with toxin-producing property 57 .Bacillus cereus was isolated from drinking water treatment plant in China and antimicrobial susceptibility testing revealed that it was resistant to cefoxitin, penicillin tetracycline 58 , macrolidelincosamide-streptogramin (MLSB), aminoglycoside and tetracycline antibiotics 59 .Assembled MAG B.cereus from effluent water contained ARGs conferring to macrolides, beta-lactams, fosfomycin and streptogramin and may be considered as MDR strain.Genomes of members of the genera Streptococcus (AS of LOS) and Enterococcus (influent), not identified at the species level, were found to contain multiple ARGs.Most of species of these genera are opportunistic and true pathogens known for their drug resistance 60,61 .One MAG from the influent water metagenome was assigned to uncultured lineage of the family Ruminococcaceae.Members of this family are typical non-pathogenic gut inhabitants, although genomes of some strains could harbor ARGs 62 .
Three MAGs retrieved from influent wastewater microbiome (Ancrocorticia) and treated effluent water (Cyclobacteriaceae and Undibacterium) were found to contain several ARGs.However, we found no evidences about pathogenic and MDR strains in these taxa.It is possible that these environmental bacteria acquired ARGs via horizontal gene from outside their lineages.WWTPs are an ideal environment for horizontal gene transfer (HGT), since when bacteria are exposed to strong selective pressures, such as the presence of antimicrobials, the horizontal acquisition of ARGs enables genetic diversification and create the potential for rapid gains in fitness 63 .

Conclusions
• Metagenome sequencing of the raw wastewater, activated sludge and treated wastewater at two large WWTPs of the Moscow city revealed several hundreds of ARGs that could confer resistance to most commonly used classes of antibiotics.• Resistome accounted for about 0.05% of the wastewater metagenome and after wastewater treatment its share decreased by 3-4 times.• The resistomes were dominated by ARGs encoding resistance to beta-lactams, macrolides, aminoglycosides, tetracycline, QAC, and sulfonamides.A peculiar feature of Moscow wastewater resistome was the high content of ARGs to sulfonamides and limited occurrence of resistance to streptogramins.• ARGs for macrolides and tetracyclines were removed more efficiently than ARGs for beta-lactamases.
• A comparison of wastewater resistomes from Moscow and around the world suggested that the abundance and content of ARG in wastewater depend on social, medical, and environmental factors.

Figure 1 .
Figure 1.Microbial community composition in the influent, AS and treated effluent samples according to 16S rRNA gene profiling.The composition is displayed at the phylum level.INFL, influent wastewater; AS-LOS, AS at LOS plant; CW-LOS, treated effluent at LOS plant; AS-NLOS2, AS at NLOS2 plant; CW-NLOS2, treated effluent at NLOS2 plant.

Figure 3 .
Figure 3.The relative abundancies of particular ARGs in the resistomes.Only ARGs with shares greater than 1% in at least one sample are shown, all other ARGs are shown as "others".

Figure 2 .
Figure 2. ARGs identified in wastewater and AS samples categorized by drug classes.

Figure 4 .
Figure 4.The relative abundancies of ARGs in the resistomes categorized by drug classes.

Table 1 .
Main parameters of influent, effluent and activated sludge in LOS and NLOS2 wastewater treatment plants.NA, not analyzed; * data for AS.